- Email: [email protected]
Structure–function relationships of autotaxin, a secreted lysophospholipase D
Advances in Biological Regulation 53 (2013) 112–117
Contents lists available at SciVerse ScienceDirect
Advances in Biological Regulation journal homepage: www.elsevier.com/locate/jbior
Structure–function relationships of autotaxin, a secreted lysophospholipase D Jens Hausmann a, Anastassis Perrakis a, Wouter H. Moolenaar b, * a b
Division of Biochemistry, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
a b s t r a c t Autotaxin (ATX or ENPP2) is an ectonucleotide pyrophosphatase/ phosphodiesterase (ENPP) that functions as a secreted lysophospholipase D to produce the multifunctional lipid mediator lysophosphatidic acid (LPA) from more complex lysophospholipids. LPA acts on distinct G protein-coupled receptors thereby activating multiple signaling cascades and cellular responses. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes, ranging from vascular and neural development to lymphocyte homing, fibrosis and cancer. Despite much progress in understanding LPA receptor signaling, the precise mode of action of ATX has long remained elusive due to the lack of structural data. In particular, it has been unclear what makes ATX a unique lysophospholipase D and how the enzyme is targeted to LPA-responsive cells. Recent structural studies have begun to clarify these issues. Here we discuss new insights and inferences from the ATX structure. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Autotaxin (ATX or ENPP2) is member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, comprising seven members with structurally related catalytic domains. The ENPPs hydrolyze phosphodiester or pyrophosphate bonds in various substrates, including nucleoside triphosphates, lysophospholipids and choline phosphate esters (Stefan et al., 2005). ATX/ENPP2 is a secreted lysophospholipase D (lysoPLD) that converts lysophosphatidylcholine (LPC) into the lipid
* Corresponding author. Tel.: þ31 20 512 1971. E-mail address: [email protected] (W.H. Moolenaar). 2212-4926/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbior.2012.09.010
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
113
mediator lysophosphatidic acid (LPA) (van Meeteren and Moolenaar, 2007). LPA, a major serum constituent, acts on six distinct G protein-coupled receptors leading to activation of multiple signaling cascades (Choi et al., 2010; Chun et al., 2010; Moolenaar et al., 2004) (Fig. 1). This results in diverse biological outcomes, depending on LPA receptor expression patterns and tissue context. Cellular responses to LPA include the stimulation of cell migration, proliferation and survival. Studies in mice have revealed key roles of the ATX-LPA signaling axis in a perplexing variety of physiological and pathological processes, including vascular and neural development (Fotopoulou et al., 2010; Tanaka et al., 2006; van Meeteren et al., 2006), embryonic left-right patterning (Lai et al., 2012), lymphocyte homing (Kanda et al., 2008), cancer (David et al., 2010; Houben and Moolenaar, 2011; Liu et al., 2009), fibrosis (Tager et al., 2008), neuropathic pain (Inoue et al., 2004) and fat mass expansion (Dusaulcy et al., 2011). From both a structural and evolutionary point of view, ATX and its ENPP family members can be divided into two main subgroups, namely ENPP1-3 and ENPP4-7 (Fig. 2). ATX/ENPP2 and its closest relatives, ENPP1 and ENPP3, have two N-terminal somatomedin B (SMB)-like domains, a central phosphodiesterase (PDE) domain and a C-terminal nuclease (NUC)-like domain. The second subgroup (ENPP4-7) has only the PDE domain in common. ATX/NPP2 is a secreted protein, while the other ENPPs are transmembrane proteins, either type-I (ENPP4-7) or type-II (ENPP1-3). ATX is by far the best characterized ENPP. The founding member, ENPP1, is an ecto-enzyme that hydrolyzes extracellular nucleotides into inorganic pyrophosphate (PPi), a key player in tissue calcification. ENPP6 and ENPP7 are choline-specific ecto-phospholipases C, likely serving catabolic roles, whereas the activities of ENPP4 and ENPP5 remain to be defined (Stefan et al., 2005).
Fig. 1. The ATX-LPA receptor signaling axis. Secreted ATX functions as a lysophospholipase D to hydrolyze lysophosphatidylcholine (LPC) into bioactive lysophosphatidic acid (LPA). LPA acts on specific G protein-coupled receptors thereby inducing multiple cellular responses, including cell migration and proliferation. The domain structure of ATX is shown in Fig. 2.
114
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
Fig. 2. The ENPP family members. Autotaxin (ENPP2) is the only lysophospholipase D in the family. Three distinct isoforms of ATX/ ENPP2 are depicted. See text for further details.
Alternative splicing gives rise to distinct isoforms of ATX (Giganti et al., 2008; van Meeteren and Moolenaar, 2007). The original ATX protein, first identified in the early 1990s as an “autocrine motility factor” for melanoma cells (Stracke et al., 1992), is termed ATXa and characterized by a 52amino acid polybasic insert in the PDE domain. The canonical and best studied isoform, termed ATXb, is identical to plasma lysoPLD and is widely expressed. A third isoform, ATXg, is mainly expressed in the central nervous system and is characterized by a 22-amino acid insertion between the PDE and NUC domain (Fig. 2). ATX can produce bioactive LPA from diverse lysophospholipid substrates, particularly LPC, the most abundant lysophospholipid in the circulation, but also from lysophosphatidylserine (LPS) and lysophosphatidylethanolamine (LPE). It thus appears that ATX does not discriminate between phospholipid headgroups. It is of note that ATX can also hydrolyze nucleotides, albeit with much lower efficiency than ENPP1. Thus, ATX/ENPP2 is a unique lysoPLD with no functional redundancy within the ENPP family. Other LPA-generating ecto/exo-phospholipases include a phosphatidic acid (PA)-specific phospholipase A1 (Inoue et al., 2011) and toxic sphingomyelinases D (van Meeteren et al., 2004) (reviewed in Moolenaar and Hla, 2012). While much has been learned about LPA receptor signaling over the last decades, many questions about ATX remained outstanding. In particular, it has been unclear what makes ATX a unique lysoPLD and how its activity is regulated. It has also remained unclear how newly produced formed LPA is specifically targeted to its cell-surface receptors. Here, we discuss new insights obtained from the ATX crystal structure. More comprehensive reviews have been published elsewhere (Moolenaar and Perrakis, 2011; Nishimasu et al., 2012). The structure of ATX The crystal structures of ATX reveal a very compact architecture (Hausmann et al., 2011; Nishimasu et al., 2011). The central PDE domain interacts with both SMB domains on one side and the NUC domain on the opposite side. The domain arrangement between the PDE and NUC domains is stabilized by four important structural features, namely (i) an interdomain disulfide bond, (ii) a “lasso loop” or linker region, (iii) an N-linked glycan chain at Asn524, and (iv) a C-terminal a-helix. The disulfide bridge between Cys413 and Cys805 connects the PDE domain directly to the NUC domain, while the lasso loop extends from the end of the PDE domain and wraps around the entire NUC domain.
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
115
The phosphodiesterase domain and substrate binding pocket The PDE domain fold is similar to that of the nucleotide pyrophosphatase from Xanthomonas axonopodis (XaNPP) (Zalatan et al., 2006). The architecture of the ATX active site around the Thr nucleophile includes two zinc ions and their coordination shell is structurally comparable to the active site of XaNPP. The metal ions coordination shell of ATX is constructed by three aspartates (Asp171, Asp311 and Asp358) and three histidines (His315, His359 and His474). All ENPP family members have conserved residues at these positions. The Thr active site residue is conserved in ENPP1-5 and ENPP7, while ENPP6 has a serine as catalytic residue, a structural common feature of other members of the alkaline phosphatase superfamily. The hydrophilic binding groove next to the active site is also a structurally conserved element between ATX and XaNPP. In XaNPP, this groove binds the ribose and base moieties of nucleotides (Zalatan et al., 2006). In ATX, however, it accommodates the phospholipid glycerol backbone. The shallow groove extends into a unique hydrophobic pocket, about 15 A in depth, which originates from a deletion of an 18-amino acid stretch that is specific to ATX (Fig. 3). The absence of this deletion in other ENPP family members strongly suggests that these enzymes lack a hydrophobic pocket. ATX in complex with diverse LPA species (different acyl chain length and saturations) shows that the hydrophobic pocket binds the acyl chain in distinct conformations (Nishimasu et al., 2011). LPAs with saturated alkyl chains (e.g. 16:0, 18:0) bind in the hydrophobic pocket in a more elongated fashion, whereas LPA with unsaturated chains (e.g. 18:1, 18:3 and 22:6) adopts a more bent conformation, most likely due to the rigidity the carbon double bond(s). In case of LPA(22:6), the aliphatic chain is even positioned in a U-shaped conformation in the hydrophobic pocket (Nishimasu et al., 2011). A boronatebased small-molecule inhibitor of ATX, termed HA155 (Albers et al., 2010), also fits into the pocket and targets the Thr nucleophile (Hausmann et al., 2011). It will be interesting to understand the lipid
Fig. 3. The structure of ATX shown as a surface representation. Distinct domains are colored according to the scheme in Fig. 2. Lower panels: the hydrophobic lipid binding pocket and the open tunnel in the catalytic domain are depicted as close-ups. The Thr nucleophile is colored in red. The images were generated using PyMOL (www.pymol.org) (PDB entry: 2XR9). See text for further details.
116
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
substrate binding properties of ENPP6 and ENPP7, as both enzymes are expected to lack a hydrophobic pocket while still being able to hydrolyze lipid substrates like LPC. The open tunnel An unexpected and intriguing feature of the ATX structure is a tunnel that spans from the active site location to the opposite side of ATX (Fig. 3). It is formed from the interaction between the SMB1 and the PDE domain (Hausmann et al., 2011; Nishimasu et al., 2011). This tunnel connects to the hydrophobic pocket and the catalytic site, thereby forming a “T-junction”. Residual electron density in the tunnel was detected. However, this density could not be unambiguously modeled, although circumstantial evidence suggest that the tunnel may function as an LPA binding site and, by inference, a product release site. It is equally well possible, however, that the tunnel serves as a lysophospholipid substrate entry site or, else, as a binding site for a yet unidentified lipid. We are currently examining the possible function(s) of this unique tunnel in further detail. The SMB domains and cell-surface binding A peculiar feature of ATX and its closest relatives, NPP1 and NPP3, are the tandem C-terminal SMB domains (SMB1 and SMB2). SMB domains are relatively small and cysteine-rich domains, known to mediate protein-protein interactions. In ATX, the SMB1 domain binds to the PDE domain with a similar surface as the SMB domain of vitronectin binds to the plasminogen activator inhibitor-1 (PAI-1) and the urokinase-type plasminogen activator. More interestingly, the SMB2 domain binds to b3 integrins, which provides a mechanism for ATX to bind to target cells, delivering LPA close to its cognate receptors (Hausmann et al., 2011). ATX contains an RGD motif in the SMB2 domain, but mutation to AGA did not affect the binding to platelet integrins (Hausmann et al., 2011). Alanine mutations of Glu109 or His119 show a significantly lower binding of ATX to activated platelets and CHO cells overexpressing the b3 subunit (Fulkerson et al., 2011). Precisely how SMB2 binds to integrins remains to be determined. One would expect that LPA production is spatially and temporally tightly regulated and integrin binding is one potential way to achieve this. Interestingly, there is evidence that SMB2integrin interaction may lead to enhanced catalytic activity (Fulkerson et al., 2011), but it remains to be examined how this is achieved at the structural level. Kanda et al. (2008) showed that ATX binds to activated lymphocytes. The binding mode is not yet clear, although a lymphocyte- and a4b1-specific 458LDV460 motif on ATX could possibly be involved. On the other hand, ATX binding to leukocytes could not be inhibited by either integrin-specific antibodies, arguing for an alternative mode of interaction (Kanda et al., 2008; Moolenaar and Perrakis, 2011). An additional or alternative scenario for ATX to bind to cells is via interaction with negatively charged heparan sulfate proteoglycans. Our preliminary evidence indeed indicates that the ATXa isoform has a high binding affinity for heparin owing to its polybasic insert (Fig. 2). It will be interesting to determine the structure of ATXa-heparin complexes to understand the binding mode in better detail. Concluding remarks ATX is arguably the most fascinating member of the ENPP family, as it is the major LPA-producing enzyme being involved in a great diversity of (patho)physiological processes. The crystal structure of ATX has answered many outstanding questions but, naturally, also has raised many new ones. Precisely how ATX activity is regulated and how the LPA product is released are questions that remain to be addressed. Furthermore, the function of the open tunnel remains enigmatic as long as it is unclear which natural molecules can be accommodated in that structure. Further structure–function analysis will undoubtedly shed more light on these issues. Conflict of interest No conflict of interest declared.
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
117
Acknowledgments Work in our laboratories is supported by the Dutch Cancer Society (KWF) and the Netherlands Organisation of Scientific Research (NWO; TOP grant to AP and WHM). References Albers HM, Dong A, van Meeteren LA, Egan DA, Sunkara M, van Tilburg EW, et al. Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation. Proc Natl Acad Sci USA 2010;107:7257–62. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, Mutoh T, et al. LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol 2010;50:157–86. Chun J, Hla T, Lynch KR, Spiegel S, Moolenaar WH. International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol Rev 2010;62:579–87. David M, Wannecq E, Descotes F, Jansen S, Deux B, Ribeiro J, et al. Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts. PLoS One 2010;5:e9741. Dusaulcy R, Rancoule C, Gres S, Wanecq E, Colom A, Guigne C, et al. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid. J Lipid Res 2011;52:1247–55. Fotopoulou S, Oikonomou N, Grigorieva E, Nikitopoulou I, Paparountas T, Thanassopoulou A, et al. ATX expression and LPA signalling are vital for the development of the nervous system. Dev Biol 2010;339:451–64. Fulkerson Z, Wu T, Sunkara M, Kooi CV, Morris AJ, Smyth SS. Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells. J Biol Chem 2011;286:34654–63. Giganti A, Rodriguez M, Fould B, Moulharat N, Coge F, Chomarat P, et al. Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization. J Biol Chem 2008;283:7776–89. Hausmann J, Kamtekar S, Christodoulou E, Day JE, Wu T, Fulkerson Z, et al. Structural basis of substrate discrimination and integrin binding by autotaxin. Nat Struct Mol Biol 2011;18:198–204. Houben AJ, Moolenaar WH. Autotaxin and LPA receptor signaling in cancer. Cancer Metastasis Rev 2011;30:557–65. Inoue A, Arima N, Ishiguro J, Prestwich GD, Arai H, Aoki J. LPA-producing enzyme PA-PLAalpha regulates hair follicle development by modulating EGFR signalling. EMBO J 2011;30:4248–60. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H. Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling. Nat Med 2004;10:712–8. Kanda H, Newton R, Klein R, Morita Y, Gunn MD, Rosen SD. Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs. Nat Immunol 2008;9:415–23. Lai SL, Yao WL, Tsao KC, Houben AJ, Albers HM, Ovaa H, et al. Autotaxin-Lpar3 signaling regulates Kupffer’s vesicle formation and left-right asymmetry in zebrafish. Development 2012. Liu S, Umezu-Goto M, Murph M, Lu Y, Liu W, Zhang F, et al. Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell 2009;15:539–50. van Meeteren LA, Frederiks F, Giepmans BN, Pedrosa MF, Billington SJ, Jost BH, et al. Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine. J Biol Chem 2004;279:10833–6. van Meeteren LA, Moolenaar WH. Regulation and biological activities of the autotaxin-LPA axis. Prog Lipid Res 2007;46:145–60. van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, van Rooijen MA, Pradere JP, et al. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Mol Cell Biol 2006;26:5015–22. Moolenaar WH, Hla T. SnapShot: bioactive lysophospholipids. Cell 2012;148:378. Moolenaar WH, Perrakis A. Insights into autotaxin: how to produce and present a lipid mediator. Nat Rev Mol Cell Biol 2011;12: 674–9. Moolenaar WH, van Meeteren LA, Giepmans BN. The ins and outs of lysophosphatidic acid signaling. Bioessays 2004;26:870–81. Nishimasu H, Ishitani R, Aoki J, Nureki O. A 3D view of autotaxin. Trends Pharmacol Sci 2012;33:138–45. Nishimasu H, Okudaira S, Hama K, Mihara E, Dohmae N, Inoue A, et al. Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. Nat Struct Mol Biol 2011;18:205–12. Stefan C, Jansen S, Bollen M. NPP-type ectophosphodiesterases: unity in diversity. Trends Biochem Sci 2005;30:542–50. Stracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, Schiffmann E, et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem 1992;267:2524–9. Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med 2008;14:45–54. Tanaka M, Okudaira S, Kishi Y, Ohkawa R, Iseki S, Ota M, et al. Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid. J Biol Chem 2006;281:25822–30. Zalatan JG, Fenn TD, Brunger AT, Herschlag D. Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: implications for mechanism and evolution. Biochemistry 2006;45:9788–803.
Contents lists available at SciVerse ScienceDirect
Advances in Biological Regulation journal homepage: www.elsevier.com/locate/jbior
Structure–function relationships of autotaxin, a secreted lysophospholipase D Jens Hausmann a, Anastassis Perrakis a, Wouter H. Moolenaar b, * a b
Division of Biochemistry, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, The Netherlands
a b s t r a c t Autotaxin (ATX or ENPP2) is an ectonucleotide pyrophosphatase/ phosphodiesterase (ENPP) that functions as a secreted lysophospholipase D to produce the multifunctional lipid mediator lysophosphatidic acid (LPA) from more complex lysophospholipids. LPA acts on distinct G protein-coupled receptors thereby activating multiple signaling cascades and cellular responses. The ATX-LPA signaling axis is implicated in a remarkably wide variety of physiological and pathological processes, ranging from vascular and neural development to lymphocyte homing, fibrosis and cancer. Despite much progress in understanding LPA receptor signaling, the precise mode of action of ATX has long remained elusive due to the lack of structural data. In particular, it has been unclear what makes ATX a unique lysophospholipase D and how the enzyme is targeted to LPA-responsive cells. Recent structural studies have begun to clarify these issues. Here we discuss new insights and inferences from the ATX structure. Ó 2012 Elsevier Ltd. All rights reserved.
Introduction Autotaxin (ATX or ENPP2) is member of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family, comprising seven members with structurally related catalytic domains. The ENPPs hydrolyze phosphodiester or pyrophosphate bonds in various substrates, including nucleoside triphosphates, lysophospholipids and choline phosphate esters (Stefan et al., 2005). ATX/ENPP2 is a secreted lysophospholipase D (lysoPLD) that converts lysophosphatidylcholine (LPC) into the lipid
* Corresponding author. Tel.: þ31 20 512 1971. E-mail address: [email protected] (W.H. Moolenaar). 2212-4926/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jbior.2012.09.010
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
113
mediator lysophosphatidic acid (LPA) (van Meeteren and Moolenaar, 2007). LPA, a major serum constituent, acts on six distinct G protein-coupled receptors leading to activation of multiple signaling cascades (Choi et al., 2010; Chun et al., 2010; Moolenaar et al., 2004) (Fig. 1). This results in diverse biological outcomes, depending on LPA receptor expression patterns and tissue context. Cellular responses to LPA include the stimulation of cell migration, proliferation and survival. Studies in mice have revealed key roles of the ATX-LPA signaling axis in a perplexing variety of physiological and pathological processes, including vascular and neural development (Fotopoulou et al., 2010; Tanaka et al., 2006; van Meeteren et al., 2006), embryonic left-right patterning (Lai et al., 2012), lymphocyte homing (Kanda et al., 2008), cancer (David et al., 2010; Houben and Moolenaar, 2011; Liu et al., 2009), fibrosis (Tager et al., 2008), neuropathic pain (Inoue et al., 2004) and fat mass expansion (Dusaulcy et al., 2011). From both a structural and evolutionary point of view, ATX and its ENPP family members can be divided into two main subgroups, namely ENPP1-3 and ENPP4-7 (Fig. 2). ATX/ENPP2 and its closest relatives, ENPP1 and ENPP3, have two N-terminal somatomedin B (SMB)-like domains, a central phosphodiesterase (PDE) domain and a C-terminal nuclease (NUC)-like domain. The second subgroup (ENPP4-7) has only the PDE domain in common. ATX/NPP2 is a secreted protein, while the other ENPPs are transmembrane proteins, either type-I (ENPP4-7) or type-II (ENPP1-3). ATX is by far the best characterized ENPP. The founding member, ENPP1, is an ecto-enzyme that hydrolyzes extracellular nucleotides into inorganic pyrophosphate (PPi), a key player in tissue calcification. ENPP6 and ENPP7 are choline-specific ecto-phospholipases C, likely serving catabolic roles, whereas the activities of ENPP4 and ENPP5 remain to be defined (Stefan et al., 2005).
Fig. 1. The ATX-LPA receptor signaling axis. Secreted ATX functions as a lysophospholipase D to hydrolyze lysophosphatidylcholine (LPC) into bioactive lysophosphatidic acid (LPA). LPA acts on specific G protein-coupled receptors thereby inducing multiple cellular responses, including cell migration and proliferation. The domain structure of ATX is shown in Fig. 2.
114
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
Fig. 2. The ENPP family members. Autotaxin (ENPP2) is the only lysophospholipase D in the family. Three distinct isoforms of ATX/ ENPP2 are depicted. See text for further details.
Alternative splicing gives rise to distinct isoforms of ATX (Giganti et al., 2008; van Meeteren and Moolenaar, 2007). The original ATX protein, first identified in the early 1990s as an “autocrine motility factor” for melanoma cells (Stracke et al., 1992), is termed ATXa and characterized by a 52amino acid polybasic insert in the PDE domain. The canonical and best studied isoform, termed ATXb, is identical to plasma lysoPLD and is widely expressed. A third isoform, ATXg, is mainly expressed in the central nervous system and is characterized by a 22-amino acid insertion between the PDE and NUC domain (Fig. 2). ATX can produce bioactive LPA from diverse lysophospholipid substrates, particularly LPC, the most abundant lysophospholipid in the circulation, but also from lysophosphatidylserine (LPS) and lysophosphatidylethanolamine (LPE). It thus appears that ATX does not discriminate between phospholipid headgroups. It is of note that ATX can also hydrolyze nucleotides, albeit with much lower efficiency than ENPP1. Thus, ATX/ENPP2 is a unique lysoPLD with no functional redundancy within the ENPP family. Other LPA-generating ecto/exo-phospholipases include a phosphatidic acid (PA)-specific phospholipase A1 (Inoue et al., 2011) and toxic sphingomyelinases D (van Meeteren et al., 2004) (reviewed in Moolenaar and Hla, 2012). While much has been learned about LPA receptor signaling over the last decades, many questions about ATX remained outstanding. In particular, it has been unclear what makes ATX a unique lysoPLD and how its activity is regulated. It has also remained unclear how newly produced formed LPA is specifically targeted to its cell-surface receptors. Here, we discuss new insights obtained from the ATX crystal structure. More comprehensive reviews have been published elsewhere (Moolenaar and Perrakis, 2011; Nishimasu et al., 2012). The structure of ATX The crystal structures of ATX reveal a very compact architecture (Hausmann et al., 2011; Nishimasu et al., 2011). The central PDE domain interacts with both SMB domains on one side and the NUC domain on the opposite side. The domain arrangement between the PDE and NUC domains is stabilized by four important structural features, namely (i) an interdomain disulfide bond, (ii) a “lasso loop” or linker region, (iii) an N-linked glycan chain at Asn524, and (iv) a C-terminal a-helix. The disulfide bridge between Cys413 and Cys805 connects the PDE domain directly to the NUC domain, while the lasso loop extends from the end of the PDE domain and wraps around the entire NUC domain.
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
115
The phosphodiesterase domain and substrate binding pocket The PDE domain fold is similar to that of the nucleotide pyrophosphatase from Xanthomonas axonopodis (XaNPP) (Zalatan et al., 2006). The architecture of the ATX active site around the Thr nucleophile includes two zinc ions and their coordination shell is structurally comparable to the active site of XaNPP. The metal ions coordination shell of ATX is constructed by three aspartates (Asp171, Asp311 and Asp358) and three histidines (His315, His359 and His474). All ENPP family members have conserved residues at these positions. The Thr active site residue is conserved in ENPP1-5 and ENPP7, while ENPP6 has a serine as catalytic residue, a structural common feature of other members of the alkaline phosphatase superfamily. The hydrophilic binding groove next to the active site is also a structurally conserved element between ATX and XaNPP. In XaNPP, this groove binds the ribose and base moieties of nucleotides (Zalatan et al., 2006). In ATX, however, it accommodates the phospholipid glycerol backbone. The shallow groove extends into a unique hydrophobic pocket, about 15 A in depth, which originates from a deletion of an 18-amino acid stretch that is specific to ATX (Fig. 3). The absence of this deletion in other ENPP family members strongly suggests that these enzymes lack a hydrophobic pocket. ATX in complex with diverse LPA species (different acyl chain length and saturations) shows that the hydrophobic pocket binds the acyl chain in distinct conformations (Nishimasu et al., 2011). LPAs with saturated alkyl chains (e.g. 16:0, 18:0) bind in the hydrophobic pocket in a more elongated fashion, whereas LPA with unsaturated chains (e.g. 18:1, 18:3 and 22:6) adopts a more bent conformation, most likely due to the rigidity the carbon double bond(s). In case of LPA(22:6), the aliphatic chain is even positioned in a U-shaped conformation in the hydrophobic pocket (Nishimasu et al., 2011). A boronatebased small-molecule inhibitor of ATX, termed HA155 (Albers et al., 2010), also fits into the pocket and targets the Thr nucleophile (Hausmann et al., 2011). It will be interesting to understand the lipid
Fig. 3. The structure of ATX shown as a surface representation. Distinct domains are colored according to the scheme in Fig. 2. Lower panels: the hydrophobic lipid binding pocket and the open tunnel in the catalytic domain are depicted as close-ups. The Thr nucleophile is colored in red. The images were generated using PyMOL (www.pymol.org) (PDB entry: 2XR9). See text for further details.
116
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
substrate binding properties of ENPP6 and ENPP7, as both enzymes are expected to lack a hydrophobic pocket while still being able to hydrolyze lipid substrates like LPC. The open tunnel An unexpected and intriguing feature of the ATX structure is a tunnel that spans from the active site location to the opposite side of ATX (Fig. 3). It is formed from the interaction between the SMB1 and the PDE domain (Hausmann et al., 2011; Nishimasu et al., 2011). This tunnel connects to the hydrophobic pocket and the catalytic site, thereby forming a “T-junction”. Residual electron density in the tunnel was detected. However, this density could not be unambiguously modeled, although circumstantial evidence suggest that the tunnel may function as an LPA binding site and, by inference, a product release site. It is equally well possible, however, that the tunnel serves as a lysophospholipid substrate entry site or, else, as a binding site for a yet unidentified lipid. We are currently examining the possible function(s) of this unique tunnel in further detail. The SMB domains and cell-surface binding A peculiar feature of ATX and its closest relatives, NPP1 and NPP3, are the tandem C-terminal SMB domains (SMB1 and SMB2). SMB domains are relatively small and cysteine-rich domains, known to mediate protein-protein interactions. In ATX, the SMB1 domain binds to the PDE domain with a similar surface as the SMB domain of vitronectin binds to the plasminogen activator inhibitor-1 (PAI-1) and the urokinase-type plasminogen activator. More interestingly, the SMB2 domain binds to b3 integrins, which provides a mechanism for ATX to bind to target cells, delivering LPA close to its cognate receptors (Hausmann et al., 2011). ATX contains an RGD motif in the SMB2 domain, but mutation to AGA did not affect the binding to platelet integrins (Hausmann et al., 2011). Alanine mutations of Glu109 or His119 show a significantly lower binding of ATX to activated platelets and CHO cells overexpressing the b3 subunit (Fulkerson et al., 2011). Precisely how SMB2 binds to integrins remains to be determined. One would expect that LPA production is spatially and temporally tightly regulated and integrin binding is one potential way to achieve this. Interestingly, there is evidence that SMB2integrin interaction may lead to enhanced catalytic activity (Fulkerson et al., 2011), but it remains to be examined how this is achieved at the structural level. Kanda et al. (2008) showed that ATX binds to activated lymphocytes. The binding mode is not yet clear, although a lymphocyte- and a4b1-specific 458LDV460 motif on ATX could possibly be involved. On the other hand, ATX binding to leukocytes could not be inhibited by either integrin-specific antibodies, arguing for an alternative mode of interaction (Kanda et al., 2008; Moolenaar and Perrakis, 2011). An additional or alternative scenario for ATX to bind to cells is via interaction with negatively charged heparan sulfate proteoglycans. Our preliminary evidence indeed indicates that the ATXa isoform has a high binding affinity for heparin owing to its polybasic insert (Fig. 2). It will be interesting to determine the structure of ATXa-heparin complexes to understand the binding mode in better detail. Concluding remarks ATX is arguably the most fascinating member of the ENPP family, as it is the major LPA-producing enzyme being involved in a great diversity of (patho)physiological processes. The crystal structure of ATX has answered many outstanding questions but, naturally, also has raised many new ones. Precisely how ATX activity is regulated and how the LPA product is released are questions that remain to be addressed. Furthermore, the function of the open tunnel remains enigmatic as long as it is unclear which natural molecules can be accommodated in that structure. Further structure–function analysis will undoubtedly shed more light on these issues. Conflict of interest No conflict of interest declared.
J. Hausmann et al. / Advances in Biological Regulation 53 (2013) 112–117
117
Acknowledgments Work in our laboratories is supported by the Dutch Cancer Society (KWF) and the Netherlands Organisation of Scientific Research (NWO; TOP grant to AP and WHM). References Albers HM, Dong A, van Meeteren LA, Egan DA, Sunkara M, van Tilburg EW, et al. Boronic acid-based inhibitor of autotaxin reveals rapid turnover of LPA in the circulation. Proc Natl Acad Sci USA 2010;107:7257–62. Choi JW, Herr DR, Noguchi K, Yung YC, Lee CW, Mutoh T, et al. LPA receptors: subtypes and biological actions. Annu Rev Pharmacol Toxicol 2010;50:157–86. Chun J, Hla T, Lynch KR, Spiegel S, Moolenaar WH. International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature. Pharmacol Rev 2010;62:579–87. David M, Wannecq E, Descotes F, Jansen S, Deux B, Ribeiro J, et al. Cancer cell expression of autotaxin controls bone metastasis formation in mouse through lysophosphatidic acid-dependent activation of osteoclasts. PLoS One 2010;5:e9741. Dusaulcy R, Rancoule C, Gres S, Wanecq E, Colom A, Guigne C, et al. Adipose-specific disruption of autotaxin enhances nutritional fattening and reduces plasma lysophosphatidic acid. J Lipid Res 2011;52:1247–55. Fotopoulou S, Oikonomou N, Grigorieva E, Nikitopoulou I, Paparountas T, Thanassopoulou A, et al. ATX expression and LPA signalling are vital for the development of the nervous system. Dev Biol 2010;339:451–64. Fulkerson Z, Wu T, Sunkara M, Kooi CV, Morris AJ, Smyth SS. Binding of autotaxin to integrins localizes lysophosphatidic acid production to platelets and mammalian cells. J Biol Chem 2011;286:34654–63. Giganti A, Rodriguez M, Fould B, Moulharat N, Coge F, Chomarat P, et al. Murine and human autotaxin alpha, beta, and gamma isoforms: gene organization, tissue distribution, and biochemical characterization. J Biol Chem 2008;283:7776–89. Hausmann J, Kamtekar S, Christodoulou E, Day JE, Wu T, Fulkerson Z, et al. Structural basis of substrate discrimination and integrin binding by autotaxin. Nat Struct Mol Biol 2011;18:198–204. Houben AJ, Moolenaar WH. Autotaxin and LPA receptor signaling in cancer. Cancer Metastasis Rev 2011;30:557–65. Inoue A, Arima N, Ishiguro J, Prestwich GD, Arai H, Aoki J. LPA-producing enzyme PA-PLAalpha regulates hair follicle development by modulating EGFR signalling. EMBO J 2011;30:4248–60. Inoue M, Rashid MH, Fujita R, Contos JJ, Chun J, Ueda H. Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling. Nat Med 2004;10:712–8. Kanda H, Newton R, Klein R, Morita Y, Gunn MD, Rosen SD. Autotaxin, an ectoenzyme that produces lysophosphatidic acid, promotes the entry of lymphocytes into secondary lymphoid organs. Nat Immunol 2008;9:415–23. Lai SL, Yao WL, Tsao KC, Houben AJ, Albers HM, Ovaa H, et al. Autotaxin-Lpar3 signaling regulates Kupffer’s vesicle formation and left-right asymmetry in zebrafish. Development 2012. Liu S, Umezu-Goto M, Murph M, Lu Y, Liu W, Zhang F, et al. Expression of autotaxin and lysophosphatidic acid receptors increases mammary tumorigenesis, invasion, and metastases. Cancer Cell 2009;15:539–50. van Meeteren LA, Frederiks F, Giepmans BN, Pedrosa MF, Billington SJ, Jost BH, et al. Spider and bacterial sphingomyelinases D target cellular lysophosphatidic acid receptors by hydrolyzing lysophosphatidylcholine. J Biol Chem 2004;279:10833–6. van Meeteren LA, Moolenaar WH. Regulation and biological activities of the autotaxin-LPA axis. Prog Lipid Res 2007;46:145–60. van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, van Rooijen MA, Pradere JP, et al. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Mol Cell Biol 2006;26:5015–22. Moolenaar WH, Hla T. SnapShot: bioactive lysophospholipids. Cell 2012;148:378. Moolenaar WH, Perrakis A. Insights into autotaxin: how to produce and present a lipid mediator. Nat Rev Mol Cell Biol 2011;12: 674–9. Moolenaar WH, van Meeteren LA, Giepmans BN. The ins and outs of lysophosphatidic acid signaling. Bioessays 2004;26:870–81. Nishimasu H, Ishitani R, Aoki J, Nureki O. A 3D view of autotaxin. Trends Pharmacol Sci 2012;33:138–45. Nishimasu H, Okudaira S, Hama K, Mihara E, Dohmae N, Inoue A, et al. Crystal structure of autotaxin and insight into GPCR activation by lipid mediators. Nat Struct Mol Biol 2011;18:205–12. Stefan C, Jansen S, Bollen M. NPP-type ectophosphodiesterases: unity in diversity. Trends Biochem Sci 2005;30:542–50. Stracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, Schiffmann E, et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem 1992;267:2524–9. Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med 2008;14:45–54. Tanaka M, Okudaira S, Kishi Y, Ohkawa R, Iseki S, Ota M, et al. Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid. J Biol Chem 2006;281:25822–30. Zalatan JG, Fenn TD, Brunger AT, Herschlag D. Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: implications for mechanism and evolution. Biochemistry 2006;45:9788–803.